Antisense oligonucleotides (AO), triple helix-forming oligonucleotides (TFO) and sense oligonucleotides have been found to be specific inhibitors of gene expression in a large number of systems, both in vitro and in vivo (e.g., Uhlmann & Peyman, Chem. Rev., 1990, 90: 543; Milligan et al., J. Med. Chem., 1993, 36: 1923; Stein & Cheng, Science, 1993, 261: 1004; Bielinaki et al., Science, 1990, 250: 997).
One of the main problems when using naturally occurring phosphodiester (PO) oligonucleotides is their rapid degradation by various nucleolytic activities both in cells and in the cell culture medium. A variety of chemical modifications have been employed in order to stabilize oligonucleotides. Reviews on the state of the art are provided, for example, by Uhlmann & Peyman, above, and P. D. Cook (Antisense Research and Applications, Crooke and Lebleu, Eds., Chapter 9, p. 149ff, CRC Press Boca Raton 1993).
Stabilization against nucleolytic degradation can be effected by modifying or replacing the phosphate bridge, the sugar structural component or the nucleotide base, or by replacing the sugar-phosphate backbone of the oligonucleotides. Numerous modifications of the internucleoside bridge have, in particular, been described, since the phosphate bridge is the center of the nucleolytic attack. The nuclease-resistant inter-nucleoside bridges which are most frequently used are phosphorothioate (PS), methyl phosphonate (MeP) and phosphoramidate (PA) bridges. Hairpin or self-stabilized oligonucleotides, as described, for example, in Tang et al., Nucl. Acids Res., 1993, 21: 2729) represent a further option for stabilization.
An additional problem in antisense technology is that cell penetration by the oligonucleotides is often inadequate. Numerous chemical modifications have been employed to improve this situation as well. Uhlmann & Peymann, above, and P. D. Cook, above, provide reviews of the state of the art. These modifications involve, inter alia, lipophilic conjugate groups at the 5′ or 3′ end of the oligonucleotide and neutral or ionic modifications of the backbone, and also 2′ modifications on the pentofuranosyl ring. Hughes, Antisense Research and Development, 1994, 4: 211, demonstrates that the cell uptake of a 10-mer homo-G phosphorothioate is higher, by a factor of 2, than the cell uptake of the 10-mer homo-oligomeric phosphorothioate of T, A or C.
Blackburne, Nature, 1991, 350: 569, describes structures which can be assumed by G-rich oligonucleotides. In the presence of Na+ or K+, G-rich oligonucleotides which possess at least four short segments containing G residues are able to form intramolecular structures which contain so-called G quartets as the stabilizing element; these quartets comprise four guanine residues which are linked in one plane by way of Hoogsten base pairing. Several such elements are arranged one above the other. Frequently, oligonucleotides are too short to form such intramolecular structures. In these cases, G quartet structures can be formed by the association of two separate oligonucleotides.